Obesity, a condition in which an amount of body fat is abnormally higher than standard body weight, refers to a disease resulting from accumulation of surplus calories in adipose tissues of the body when calorie intake is greater than calorie expenditure. Complications caused from obesity include, for example hypertension, myocardiac infarction, varicosis, pulmonary embolism, coronary artery diseases, cerebral hemorrhage, senile dementia, Parkinson's disease, type 2 diabetes, hyperlipidemia, cerebral apoplexy, various cancers (such as uterine cancer, breast cancer, prostate cancer, colon cancer and the like), heart diseases, gall bladder diseases, sleep apnea syndrome, arthritis, infertility, venous ulcer, sudden death, fatty liver, hypertrophic cardiomyopathy (HCM), thromboembolism, esophagitis, abdominal wall hernia (Ventral Hernia), urinary incontinence, cardiovascular diseases, endocrine diseases and the like (Obesity Research Vol. 12(8), 2004, 1197-1211).
Diabetes is a systemic metabolic disorder resulting from multiple environmental and genetic factors, and refers to a condition characterized by abnormally elevated blood glucose levels due to absolute or relative deficiency of insulin in the body. Complications of diabetes includes, for example hypoglycemia, ketoacidosis, hyperosmolar coma, macrovascular complications, diabetic retinopathy, diabetic neuropathy, diabetic nephropathy and the like.
Metabolic syndromes refer to syndromes accompanied by health risk factors such as hypertriglyceridemia, hypertension, glycometabolism disorders, blood coagulation disorders and obesity. According to the ATP III criteria of the National Cholesterol Education Program (NCEP) published in 2001, individuals are diagnosed with the metabolic syndrome by the presence of three or more of the following components: 1) A waistline of 40 inches (102 cm) or more for men and 35 inches (88 cm) or more for women (central obesity as measured by waist circumference), 2) A triglyceride level above 150 mg/dl, 3) A high density lipoprotein level (HDL) less than 40 mg/dl (men) or under 50 mg/dl (women), 4) A blood pressure of 130/85 mm Hg or higher and 5) A fasting blood glucose level greater than 110 mg/dl.
Insulin resistance refers to a phenomenon wherein, even though insulin is normally secreted in the body, “supply of glucose into cells” performed by insulin does not work properly. Therefore, glucose in the blood cannot enter cells, thus causing hyperglycemia, and further, cells themselves cannot perform normal functions thereof due to a shortage of glucose, leading to the manifestation of metabolic syndrome.
The degenerative disease is the term derived from pathological findings, thus meaning the condition which is accompanied by “decreases in consumption of oxygen”, and refers to a degenerative disease wherein dysfunction of mitochondria, which is an organelle that generates energy using oxygen within the cell, is related to senescence. As examples of the degenerative disease, mention may be made of neurodegenerative disease such as Alzheimer's disease, Parkinson's disease and Huntington's disease (Korean Society of Medical Biochemistry and Molecular Biology News, 2004, 11(2), 16-22).
Diseases arising from mitochondrial dysfunction may include for example, mitochondrial swelling due to mitochondrial membrane potential malfunction, functional disorders due to oxidative stress such as by the action of active oxygen species or free radicals, functional disorders due to genetic factors, and diseases due to functional deficiency of oxidative phosphorylation mechanisms for energy production of mitochondria. Specific examples of diseases, developed by the above-mentioned pathological causes, may include multiple sclerosis, encephalomyelitis, cerebral radiculitis, peripheral neuropathy, Reye's syndrome, Friedrich's ataxia, Alpers syndrome, MELAS, migraine, psychosis, depression, seizure and dementia, paralytic episode, optic atrophy, optic neuropathy, retinitis pigmentosa, cataract, hyperaldosteronemia, hypoparathyroidism, myopathy, amyotrophy, myoglobinuria, hypotonia, myalgia, the decrease of exercise tolerance, renal tubulopathy, renal failure, hepatic failure, liver function failure, hepatomegaly, red blood cell anemia (iron-deficiency anemia), neutropenia, thrombocytopenia, diarrhea, villous atrophy, multiple vomiting, dysphagia, constipation, sensorineural hearing loss (SNHL), epilepsy, mental retardation, Alzheimer's disease, Parkinson's disease and Huntington's disease (see, for example U.S. Pat. No. 6,183,948, Korean Patent Laid-open Publication No. 2004-7005109, Journal of clinical investigation 111, 303-312, 2003, Mitochondria 74, 1188-1199, 2003, Biochimica et Biophysica acta 1658 (2004) 80-88).
The above-mentioned obesity, diabetes, metabolic syndromes, degenerative diseases and mitochondrial dysfunction-related diseases will be collectively referred to as “disease syndromes” hereinafter.
At present, the most effective way to ameliorate or fight against the conditions associated with such disease syndromes is known to be getting more exercise and losing weight, and dietary control. All of the currently effective ways of fighting against the disease syndromes have in common the fact that they facilitate energy metabolism, thus resulting in maximized expenditure of surplus energy in the body leading to prevention of energy accumulation. Effective expenditure of such surplus energy is considered a method for treating the disease syndromes. Promoting energy metabolism is most important for effective elimination of surplus energy. For this purpose, it is essential to achieve inhibition of lipogenesis, inhibition of gluconeogenesis, facilitation of glucose consumption, facilitation of fat oxidation, facilitation of biogenesis of mitochondria which is a central apparatus of energy metabolism and collective activation of factors involved in metabolic activation.
There is yet little known about targets to treat the disease-syndromes, whereas numerous target proteins or genes are known only for treating individual diseases and therefore there have been proposed some methods for the prevention or treatment of such diseases via use of the above-mentioned corresponding target proteins or genes. However, there is still a room for further significant improvement even in treatment of individual diseases such as metabolic syndromes including obesity, diabetes and the like. In spite of the fact that a great deal of studies have been conducted on treatment of diseases, there are yet no drugs available for the treatment of various diseases resulting from excess energy intake and aging.
Most of diseases including obesity, diabetes, metabolic syndromes, degenerative diseases and mitochondrial dysfunction-related diseases, i.e., large numbers of diseases including disease syndromes, stem from imbalance of energy metabolism and oxidation-reduction state. For this reason, the present invention has also employed a method of confirming the presence/absence of activation effects on AMP-activated protein kinase (AMPK), as the most fundamental primary test to confirm biological efficacy of compounds of interest on disease syndromes.
Meanwhile, once AMPK is activated, a variety of physiological events are consequently affected in the downstream of the mechanism thereof. In this regard, factors to be regulated and expression phenomena are provided as follows.
1. Glycometabolism
In muscle tissues and myocardial tissues, AMPK promotes muscle contraction and thereby facilitates intake of glucose. That is, AMPK activates GLUT 1, or induces migration of GLUT 4 to a plasma membrane, regardless of insulin action, resulting in increased glucose uptake into cells (Arch. Biochem. Biophys. 380, 347-352, 2000, J. Appl. Physiol. 91, 1073-1083, 2001). After increasing glucose uptake into cells, AMPK activates hexokinase, thereby increasing flux of glycometabolism processes and simultaneously inhibiting glycogen synthesis. It is known that in myocardial tissues under ischemic conditions, AMPK activates a phosphorylation process of 6-phosphofructo-2-kinase (PFK-2), with consequent activation of a metabolic cascade leading to increased flux of glycometabolism (Curr. Biol. 10, 1247-1255, 2000). In addition, it was confirmed that activation of AMPK in the liver inhibits release of glucose from hepatocytes, and activity of phosphoenolpyruvate carboxykinase (PEPCK) and glucose-6-phosphatase, which are gluconeogenesis enzymes, is inhibited by AMPK (Diabetes 49, 896-903, 2000). This is because AMPK independently takes part in regulation of a blood glucose level via inhibition of release of glucose from the liver, irrespective of insulin.
2. Mitochondrial Biogenesis
One important function of mitochondria is to carry out an oxidative phosphorylation process, which converts energy produced from fuel metabolites such as glucose and fatty acids into ATP. It is known that the incidence of disorders in mitochondrial functions is involved in a pathogenic mechanism of various degenerative diseases associated with senescence, such as diabetes, cardiovascular diseases, Parkinson's disease and senile dementia (Curr. Opin. Cell Biol. 15, 706-716, 2003). Peterson, et al (Science 300, 1140-1142, 2003) have suggested the possibility that deteriorated mitochondrial function is a probable pathogenic cause of insulin resistance syndrome, with reporting that oxidative phosphorylation functions of mitochondria were weakened by about 40% in the elderly. Lee, et al (Diabetes Res. Clin. Pract. 42, 161-167, 1998) have confirmed that a decrease in the content of mitochondrial DNA in the peripheral blood is initiated from before the incidence of diabetes. Biogenesis of mitochondria in muscles is known to be promoted by an adaptive reaction in which metabolic activity of oxidative phosphorylation of muscle cells is increased by chronic energy depletion and exercise. Zong, et al (Proc Natl. Acad. Sci. USA 99: 15983-15987, 2002) have revealed that, using a transgenic mouse in which AMPK was genetically inactivated, AMPK is required for mitochondrial biogenesis in skeletal muscle under conditions in which chronic energy deprivation was induced. Further, Putman, et al (J. Physiol. 551, 169-178, 2003) have demonstrated the hypothesis that AMPK in association with continuous exercise is involved in an increase of mitochondrial volume.
Meanwhile, it was confirmed that AMPK increases gene expression of a peroxisome proliferator-activated receptor gamma coactivator 1α(PGC-1α) which is known to play an important role in mitochondrial biogenesis (Endocr. Rev. 24, 78-90, 2003). Raynald, et al (Am. J. Physiol. Endocrinol. Metab. 281, 1340, 2001) have suggested that a nuclear respiratory factor-1 (NRF-1), which is a gene essential for transcription of proteins associated with a mitochondrial respiratory system as well as mitochondrial transcription and replication, plays an important role to increase oxidation capability in muscle cells in response to chronic energy stress. Therefore, NRF-1 consequently participates in an increase of mitochondrial biogenesis. In addition, it is known that enzymatic activity of citrate synthase and 3-hydroxyacyl-CoA dehydrogenase, known as being increased in conjunction with increased amounts of UCP-3 protein and mRNA thereof and increased mitochondrial volume, is increased by activation of AMPK (J. Physiol. 551, 169-178, 2003).
3. Fat Metabolism Regulation and AMPK
Upon reviewing a mechanism of AMPK participating in fat metabolism, AMPK induces phosphorylation of acetyl-CoA carboxylase, thereby resulting in inhibition of fatty acid synthesis. Therefore, AMPK is known to facilitate fatty acid oxidation, by the action of decreasing an intracellular concentration of malonyl-CoA that is an intermediate of fatty acid synthesis and is an inhibitor of carnitine palmitoyl-CoA transferase I (CPT I). CPT I is an enzyme essential for a fatty acid oxidation process wherein fatty acids enter mitochondria and are oxidized, and is known under the control of malonyl-CoA. In addition, AMPK is known to inhibit activity of HMG-CoA reductase and glycerol phosphate acyl transferase (GPAT), involved in synthesis of cholesterol and triacylglycerol, through phosphorylation (J. Biol. Chem. 277, 32571-32577, 2002, J. Appl. Physiol. 92, 2475-2482, 2002).
Meanwhile, it was found that activation of AMPK in the liver inhibits the activity of pyruvate kinase, fatty acid synthase and ACC through phosphorylation of carbohydrate-response-element-binding protein (ChREBP) (J. Biol. Chem. 277, 3829-3835, 2002). In addition, activity of sterol-regulatory-element binding protein-1 (SREBP-1), which plays an important role in differentiation of adipocytes, is also inhibited by the action of AMPK, which results then in inhibition of adipocyte differentiation.
4. Protein Synthesis Regulation and AMPK
In the protein synthesis process, AMPK inhibits synthesis of proteins via inhibition of mTOR and p70S6K by activating TSC, or AMPK inhibits translation elongation via activation of elongation factor-2 (eEF2) kinase and inactivation of eEF2 through phosphorylation thereof. It was found that eEF2 kinase is a direct substrate for AMPK (J. Biol. Chem. 278, 41970-41976, 2003).
As discussed above, AMPK is known to play a central role in energy metabolism of glucose, protein and fat in vitro and in vivo. Neil, et al (Nature drug discovery, 3(April), 340, 2004) has asserted that AMPK and Malonyl-CoA are possible targets for the treatment of metabolic syndromes, and they have also stated that patients suffering from metabolic syndromes can be characterized by insulin resistance, obesity, hypertension, dyslipidemia, and dysfunction of pancreatic beta cells, type II diabetes and manifestation of arteriosclerosis. It was hypothesized that a common feature linking these multiple abnormalities is dysregulation of AMPK/Malonyl-CoA energy level-sensing and signaling network. It was proposed that such dysregulation leads to alterations in cellular fatty-acid metabolism that in turn cause abnormal fat accumulation, cellular dysfunction and ultimately disease. Evidence is also presented that factors activating AMPK and/or reducing malonyl-CoA levels might reverse these abnormalities and syndromes or prevent incidence of these diseases.
Roger, et al (Cell, 117, 145-151, 2004) have suggested that AMPK may be a possible target to control obesity by lowering activity of hypothalamic AMPK, thereby increasing a content of malonyl-CoA and then regulating appetite for food intake.
Lee, et al (Nature medicine, 13(June), 2004) have suggested that alpha-lipoic acid can exert anti-obesity effects by suppressing hypothalamic AMPK activity, thus controlling appetite. They have also reported that alpha-lipoic acid promotes fat metabolism via activation of AMPK in muscle tissues, not hypothalamus, and alpha-lipoic acid is therapeutically effective for the treatment of obesity because it facilitates energy expenditure by activating UCP-1, particularly in adipocytes.
Diraison, et al (Diabetes 53, S84-91, 2004) have reported that activation of AMPK in pancreatic cells leads to four-fold increases in expression of the gut hormone peptide YY responsible for appetite control and thus appetite can be regulated by the action of AMPK in other tissues other than hypothalamus.
Nandakumar, et al (Progress in lipid research 42, 238-256, 2003) have proposed that, in ischemic heart diseases, AMPK would be a target to treat ischemia reperfusion injuries via regulation of fat and glucose metabolism.
Min, et al (Am. J. Physiol. Gastrointest Liver Physiol 287, G1-6, 2004) have reported that AMPK is effective for regulation of alcoholic fatty liver.
Genevieve, et al (J. Biol. Chem. 279, 20767-74, 2004) have reported that activation of AMPK inhibits activity of an iNOS enzyme that is an inflammation mediator in chronic inflammatory conditions or endotoxin shock, including obesity-related diabetes and thus AMPK will be effective for developing new medicines having a mechanism capable of enhancing insulin sensitivity. In addition, they have reported that inhibition of iNOS activity is effected by activation of AMPK, and thus this finding is clinically applicable to diseases such as septicemia, multiple sclerosis, myocardial infarction, inflammatory bowel diseases and pancreatic beta-cell dysfunction.
Zing-ping et al (FEBS Letters 443, 285-289, 1999) have reported that AMPK activates endothelial NO synthase through phosphorylation, in the presence of Ca-calmodulin in murine muscle cells and myocardial cells. This represents that AMPK is implicated in heart diseases including angina pectoris.
Javier, et al (Genes & Develop. 2004) have reported that a lifespan can be extended by limiting utilization of energy and such a prolonged lifespan is achieved in a manner that an in vivo AMP/ATP ratio is increased and therefore the α2 subunit of AMPK is activated by AMP. Therefore, they have suggested that AMPK may function as a sensor to detect the relationship between lifespan extension and energy level and insulin-like signal information.
Meanwhile, Danshen (Salvia miltiorrhiza) has been widely used as an important herbal medicine in Northeast Asia regions since ancient times, and is well-known to have excellent effects on prevention and treatment of various cardiovascular diseases. Upon focusing our attention to such therapeutic efficacy of Danshen, the inventors of the present invention have suggested that main ingredients of Danshen are superb medicinal substances capable of treating various diseases such as obesity, diabetes and metabolic syndromes. For example, see Korean Patent Nos. 2003-0099556, 2003-0099557, 2003-0099657, 2003-0099658, 2004-0036195, 2004-0036197 and 2004-0050200, assigned to the present applicant. In particular, the present inventors have revealed that main principles of Danshen including Cryptotanshinone, 15,16-Dihydrotanshinone, Tanshinone II-A, and Tanshinone I can treat metabolic syndrome diseases.
